Abstract: Acute gastroenteritis and severe diarrhea is a leading cause of preventable death in infants and young children worldwide. It ranks second to neonatal deaths as the major cause of childhood mortality. In this regard, rotavirus infection is the most important microbial causative agent, particularly in developing countries. Coherently, this study was designed to assess the prevalence of rotavirus infection among young children with acute gastroenteritis and severe diarrhea in Holy Makkah, Saudi Arabia and evaluate the specificity of Latex Agglutination Test (LAT) in detection of rotavirus infection in comparison with Reverse-Transcription Polymerase Chain Reaction (RT-PCR). During the period from March to September 2011, stool samples were collected from 100 children (of both sex and under 5 years of age) with acute gastroenteritis and severe diarrhea and admitted to the pediatric wards at the Garwal Hospital in Holly Makkah. For detection of rotavirus infection, the samples were first examined by LAT and then by RT-PCR using specific primer sequences for human rotavirus VP4 and VP7 genes. Sixteen samples of these 100 samples (16%) were positive for rotavirus by LAT. However, the genetic materials of rotavirus (VP4 and VP7 genes) were only detected in 4 samples of these 100 samples (4%) by RT-PCR amplification. The samples that tested negative with LAT were also negative with RT-PCR. Overall, this study indicated that rotavirus infection still responsible for acute gastroenteritis and severe diarrhea among children in Holy Makkah with a prevalence of 4%. In turn, this may help in assessing the success of the rotavirus vaccine in the future. In addition, this study reflects the low specificity of LAT than RT-PCR for detection of rotavirus infection.